The dopamine transporter (DAT) mediates the inactivation of released dopamine (DA) through its reuptake. The long-term goals of this research are to understand how DA accumulation and efflux are regulated by post-translatlonal modification of DAT, by association of DAT with other proteins, and by localization of DAT to specific membrane microdomains. During the previous project period we demonstrated that phosphorylation ofthe DAT N-terminus is essential for AMPH-induced DA efflux, CaMKIIa binds to the distal C-termlnus of DAT, and CaMKIIa phosphorylates serines In the distal N-terminus of DAT in vitro. The CaMKll Inhibitor KN93 reduces AMPH-induced DA efflux in cells as well as in vivo in mouse striatum. In order to develop a model system for mechanistic examination, we have established a behavioral assay for AMPH-induced DAT-mediated DA efflux in Drosophila melanogaster larvae. In larvae, inhibition of CaMKll only in DA neurons inhibits the AMPH-induced behavior, whereas expression of constitutively active CaMKll enhances AMPH-induced behavior. We have shown that the membrane raft-associated protein Flotillini (Floti) is necessary for localization of DAT in membrane rafts. Floti knockdown blunts AMPH-induced DA efflux in mouse DA neurons in primary culture and AMPH-induced behavior in D. melanogaster larvae. The precise mechanisms by which Floti modulates DAT localization and function remain unknown. Our working hypothesis is that Floti traffics DAT to a membrane raft compartment containing the necessary signaling machinery to phosphorylate the DAT N-terminus and thereby allow AMPH-induced DA efflux. We propose to: 1) characterize the relationship between Floti, DAT, and DAT-interacting proteins in membrane rafts, 2) determine the role of Floti in AMPH-induced DA efflux and behavio, and 3) determine the role of N-terminal phosphorylation of DAT and its raft localization in AMPH-induced DA efflux and behavior.These aims will be pursued in heterologous cells, in intact behaving D. melanogaster larvae, and in genetically modified mice, in a collaborative and synergistic interaction with the other PPG projects and the electrophysiological expertise of the core. RELEVANCE (See instmctions): The dopamine transporter is responsible for the rewarding properties and abuse potential of cocaine, amphetamine, and related psychostimulants. The related transporters for serotonin and norepinephrine are targets for antidepressant medications and secondary targets for psychostimulants. We strive to understand how these transporters are regulated in health and In disease.